Search

OBJECTIVES: Abiraterone acetate is a well-known anticancer drug and a steroidal derivative of progesterone for treatment of patients with hormone-refractory prostate cancer. Chemometrics-assisted reverse phase high performance liquid chromatography (RP-HPLC) development of the drug abiraterone acetate has been employed in this study using an analytical quality by design (AQbD) approach. MATERIALS AND METHODS: Drug separation was performed using a Princeton Merck-Hibar Purospher STAR (C18, 250 mm × 4.6 mm) i.d., 5 μm particle size) with ultraviolet detection at 235 nm. A Box-Behnken statistical experimental design with response surface methodology was executed for method optimization and desired chromatographic separation from its formulation with a few numbers of experimental trials. The impact of three independent variables, namely, composition of the mobile phase, pH, and flow rate, on response retention time and peak area was studied by constructing an arithmetic model from these variables. RESULTS: Optimized experimental conditions for the proposed work include the mobile phase acetonitrile and phosphate buffer (10 mM KH(2)PO(4)) (20:80 %v/v). At the concentration range of 2-100 μg/mL, a linear calibration curve was found. Recovery was performed at three concentrations and was foun to be between 98% and 102%. The 3D response surface curves revealed that mobile phase composition and flow rate were the most substantial critical factors affecting desired responses. CONCLUSION: An attempt has been made to develop and validate an economical, precise, robust, stability-indicating AQbD-based RP-HPLC method that can be employed successfully for the routine analysis of abiraterone acetate in quality control labs.

This study tests biodegradation resistance of a custom synthesized novel ethylene glycol ethyl methacrylate (EGEMA) with ester bond linkages that are external to the central polymer backbone when polymerized. Ethylene glycol dimethacrylate (EGDMA) with internal ester bond linkages and EGEMA discs were prepared in a polytetrafluoroethylene (PTFE) mold using 40 μl macromer and photo/co-initiator mixture cured for 40 s at 1000 mW/cm(2). The discs were stored in the constant presence of Streptococcus mutans (S. mutans) in Todd Hewitt Yeast + Glucose (THYE+G) media up to 9 weeks ([Formula: see text] for each macromer type) and physical/mechanical properties were assessed. Initial measurements EGEMA versus EGDMA polymer discs showed equivalent degree of conversion (45.69% ± 2.38 vs. 46.79% ± 4.64), diametral tensile stress (DTS; 8.12± 2.92 MPa vs. 6.02 ± 1.48 MPa), and low subsurface optical defects (0.41% ± 0.254% vs. 0.11% ± 0.074%). The initial surface wettability (contact angle) was slightly higher [Formula: see text] for EGEMA (62.02° ± 3.56) than EGDMA (53.86° ± 5.61°). EGDMA showed higher initial Vicker’s hardness than EGEMA (8.03 ± 0.88 HV vs. 5.93 ± 0.69 HV; [Formula: see text]). After 9 weeks of S. mutans exposure, EGEMA ([Formula: see text]) showed higher resistance to biodegradation effects with a superior DTS than EGDMA ([Formula: see text]) [Formula: see text]. Visible and scanning electron microscopy images of EGEMA show less surface cracking and defects than EGDMA. EGDMA had higher loss of material (18.9% vs. 8.5%, [Formula: see text]), relative changes to fracture toughness (92.5% vs. 49.2%, [Formula: see text]) and increased water sorption (6.1% vs. 1.9%, [Formula: see text]) compared to EGEMA discs. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to bacterial degradation effects than an internal ester group linkage design methacrylate.

Parvalbumin-expressing fast-spiking interneurons (PV-INs) within feedforward microcircuits in the nucleus accumbens (NAc) coordinate goal-directed motivational behavior. Feedforward inhibition of medium spiny projection neurons (MSNs) is initiated by glutamatergic input from corticolimbic brain structures. While corticolimbic synapses onto MSNs are targeted by the psychostimulant, cocaine, it remains unknown whether cocaine also exerts acute neuromodulatory actions at collateralizing synapses onto PV-INs. Using whole-cell patch-clamp electrophysiology, optogenetics, and pharmacological tools in transgenic reporter mice, we found that cocaine decreases thalamocortical glutamatergic drive onto PV-INs by engaging a monoamine-independent mechanism. This mechanism relies on postsynaptic sigma-1 (σ1) activity, leading to the mobilization of intracellular Ca2+ stores that trigger retrograde endocannabinoid signaling at presynaptic type-1 cannabinoid receptors (CB1R). Cocaine-evoked CB1R activity occludes the expression of CB1R-dependent long-term depression (LTD) at this synaptic locus. These findings provide evidence that acute cocaine exposure targets feedforward microcircuits in the NAc and extend existing models of cocaine action on mesolimbic reward circuits.

Semiconducting metal oxide-based gas sensors have inadequate selectivity as they are responsive toward a variety of gases. Here, we report the implementation of gas sensing kinetic analysis of the sensor to identify the tested volatile organic compounds (VOCs) (2-propanol, formaldehyde, methanol, and toluene) precisely. A single chemiresistive sensor was employed having tin oxide-based hollow spheres as the sensing material, which were obtained by chemical synthesis. The gas sensing measurements were conducted in a dynamic manner where the sensor displayed excellent response with high sensitivity. Eley-Rideal model was adopted to obtain the kinetic properties of the gas sensing phenomenon through theoretical fitting of response transient curves and their corresponding kinetic parameters. The calculated characteristic kinetic properties were further examined to discriminate among different VOCs. The approach of using gas sensing kinetic analysis for multiple gas discrimination is an attractive solution to mitigate the problem of cross-sensitivity for resistive gas sensors.

The announcement of the rare feat of ‘unboiling an egg’ in 2015 by biochemists from University of California, Irvine and Australia was more than just a chemistry demonstration. Pulling apart tangled proteins and allowing them to refold, the new technique not only prevented ‘misfolding’, thus also eliminating the chances for formation of useless products, but also speeded up the process by a factor of thousands. This technology promised transforming industrial and research production of proteins – affecting cheese flavouring and cancer treatment alike. A survey of 50 odd students in 2020, willing to enroll for an online Food-Science Workshop revealed, 72% did not believe that such an act like ‘unboiling an egg’ was possible, leave alone its being prevalent and essential. Delving into the nuances of this ‘egg-unboiling’ technology whilst contrasting it with ‘egg-unfrying’, our Workshop Incredible Edibles introduced sustainable-science in the guise of food-science. It is how lysozyme, urea, along with Maillard reaction, amino acids and sugars came together in the discussions that determined the gentle transition of these non-believers to hard-core fanatics of chemistry −76% participants admitted to having “discovered a whole new way to look at food” in the post-session survey. This article shall elucidate these results.

The calmodulin (CaM) activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2 K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF-2 K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution-state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+ -CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C-lobe surface, complementary to the one involved in recognizing the calmodulin-binding domain (CBD) of TR, provides a secondary TR-interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+ -CaM•TR complex. Solution NMR data suggest that CaMH107K , which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild-type CaM. This article is protected by copyright. All rights reserved.

Conventional Austempered Ductile Irons (ADI) are lacking in adequate wear resistance. Introduction of hard alloy carbide phase in the microstructure by strong carbide formers imparts wear resistanc...

Background: Quality by design-based software’s in analytical research and development normally encompasses multiple objectives. For decades, this task has been attempted through trial and error, supplemented with the previous experience, knowledge, and wisdom of analytical researchers. Objective: The study analyzes the current QbD-assisted software’s, such as design-experts, minitab, fusion product development, etc., and its broad implementations in an analytical research and development. Methods: The traditional approach may fails to meet the intended purpose by trial and error procedure during analytical research and development. However, modern scientific technology is equipped with highly advanced features associated with the software of the QbD paradigm. The impact and interactions between the critical process variables and critical method attributes such as resolution, tailing, etc. can be well understood by the screening, optimization, and robustness studies based on the principles of experimental design. Results: The design of experiments assimilate statistical multi-variate analysis instead of one factor at a time approach. This also provides a prominent, most reliable quality output, which is also essential for getting highly robust method as well as to obtain homogenous product development. Conclusion: The present review, critically discussed about the various QbD based multivariate software and their applications in drug development and analytical research.

The activated hepatic stellate cells (HSCs) are the major cells that secrete the ECM proteins and drive the pathogenesis of fibrosis in chronic liver disease. Targeting of HSCs by modulating their activation and proliferation has emerged as a promising approach in the development of anti-fibrotic therapy. Sorafenib, a multi-kinase inhibitor has shown anti-fibrotic properties by inhibiting the survival and proliferation of HSCs. In present study we investigated sorafenib induced cytoplasmic vacuolation mediated decreased cell viability of HSCs in dose and time dependent manner. In this circumstance, sorafenib induces ROS and ER stress in HSCs without involvement of autophagic signals. The protein synthesis inhibitor cycloheximide treatment significantly decreased the sorafenib-induced cytoplasmic vacuolation with increasing cell viability. Antioxidant human serum albumin influences the viability of HSCs by reducing sorafenib induced vacuolation and cell death. However, neither caspase inhibitor Z-VAD-FMK nor autophagy inhibitor chloroquine could rescue the HSCs from sorafenib-induced cytoplasmic vacuolation and cell death. Using TEM and ER organelle tracker, we conclude that the cytoplasmic vacuoles are due to ER dilation. Sorafenib treatment induces calreticulin and GPR78, and activates IRE1α-XBP1s axis of UPR pathway, which eventually trigger the non-apoptotic cell death in HSCs. This study provides a notable mechanistic insight into the ER stress directed non-apoptotic cell death with future directions for the development of efficient anti-fibrotic therapeutic strategies.

The modification of simultaneously existing multiple oxidation states in host lattice cations via the introduction of dopants has been reported for NiO- and Co3O4-based gas-sensing materials. However, SnO2, a widely used material for chemiresistive gas sensing has never been reported with simultaneous presence of Sn2+ and Sn4+ states, in both the absence and presence of dopants. In this work, we demonstrated how antimony doping in a 3+ state triggers the generation of cationic bivalency in tin oxide-based gas sensors, and it is the quantitative presence of unstable Sn2+ species that determines the fate of SO2-sensing responses by antimony-doped tin oxide gas sensors. While the Sn2+ content in Sn0.856Sb0.144O2 is 1.2 times less than that of Sn0.957Sb0.043O2, the SO2 sensing response in the former is 1.2 times more than that in the latter. Greater antimony content in Sn0.856Sb0.144O2 also leads to the generation of additional trap states that result in sequential return of electrons back into the valence band. The reversibility of Sn2+ ↔ Sn4+ during SO2 adsorption and desorption brings out a new dimension of SnO2-based chemiresistors besides those already existing.

Flexible metal-organic frameworks (MOFs) hold great promise as smart materials for specific applications such as gas separation. These materials undergo interesting structural changes in response to guest molecules, which is often associated with unique adsorption behavior not possible for rigid MOFs. Understanding the dynamic behavior of flexible MOFs is crucial yet challenging as it involves weak host-guest interactions and subtle structural transformation not only at the atomic/molecular level but also in a nonsteady state. We report here an in-depth study on the adsorbate- and temperature-dependent adsorption in a flexible MOF by crystallizing atomic gases into its pores. Mn(ina)2 shows an interesting temperature-dependent response toward noble gases. Its nonmonotonic, temperature-dependent adsorption profile results in an uptake maximum at a temperature threshold, a phenomenon that is unusual. Full characterization of Xe-loaded MOF structures is performed by in situ single-crystal and synchrotron X-ray diffraction, IR spectroscopy, and molecular modeling. The X-ray diffraction analysis offers a detailed explanation into the dynamic structural transformation and provides a convincing rationalization of the unique adsorption behavior at the molecular scale. The guest and temperature dependence of the structural breathing gives rise to an intriguing reverse of Xe/Kr adsorption selectivity as a function of temperature. The presented work may provide further understanding of the adsorption behavior of noble gases in flexible MOF structures.

Growth cone - mediated axonal outgrowth and accurate synaptic targeting are central to brain morphogenesis. Translocation of the growth cone necessitates mechanochemical regulation of cell - extracellular matrix interactions and the generation of propulsive traction forces onto the growth environment. However, the molecular mechanisms subserving force generation by growth cones remain poorly characterized. The formin family member, Fmn2, has been identified earlier as a regulator of growth cone motility. Here, we explore the mechanisms underlying Fmn2 function in the growth cone. Evaluation of multiple components of the adhesion complexes suggests that Fmn2 regulates point contact stability. Analysis of F-actin retrograde flow reveals that Fmn2 functions as a clutch molecule and mediates the coupling of the actin cytoskeleton to the growth substrate, via point contact adhesion complexes. Using traction force microscopy, we show that the Fmn2-mediated clutch function is necessary for the generation of traction stresses by neurons. Our findings suggest that Fmn2, a protein associated with neurodevelopmental and neurodegenerative disorders, is a key regulator of a molecular clutch activity and consequently motility of neuronal growth cones.

Directed synthesis promises control over architecture and function of framework materials. In practice, however, designing such syntheses requires a detailed understanding of the multistep pathways of framework formations, which remain elusive. By identifying intermediate coordination complexes, this study provides insights into the complex role of a structure-directing agent (SDA) in the synthetic realization of a promising material. Specifically, a novel molecular intermediate was observed in the formation of an indium zeolitic metal-organic framework (ZMOF) with a sodalite topology. The role of the imidazole SDA was revealed by time-resolved in situ powder X-ray diffraction (XRD) and small-angle X-ray scattering (SAXS).

Recently, green synthesis of silver/silver chloride nanoparticles (Ag/AgCl-NPs) has gained a lot of interest because of the usage of natural resources, rapidness, eco-friendliness, and benignancy. Several researchers reported that silver-based biogenic NPs have both antimicrobial and anticancer properties. In the present study, Ag/AgCl-NPs were synthesized from Zizyphus mauritiana fruit extract, and their antibacterial, antifungal, and antiproliferative mechanisms against human MCF-7 cell lines were evaluated. Synthesis of Ag/AgCl-NPs from the Z. mauritiana fruit extract was confirmed by the changes of color and a peak of the UV–visible spectrum at 428 nm. The nanoparticles were characterized by transmission electron microscopy, energy dispersive X-ray, X-ray powder diffraction, thermal gravimetric analysis, atomic force microscope, and Fourier transform infrared. Antibacterial activity was checked against four pathogenic bacteria and two fungi. Cytotoxicity was checked against human breast cancer cell line (MCF-7) and mice Ehrlich ascites carcinoma (EAC) cells by MTS assay and clonogenicity assay. Cell morphology of the control and nanoparticle-treated MCF-7 cells were checked by Hoechst 33342, YF488-Annexin V, and caspase-3 substrates. The level of reactive oxygen species (ROS) was studied by using 2′,7′-dichlorofluorescein-diacetate staining. Real-time polymerase chain reaction was used for gene expression. Synthesized nanoparticles were heat stable cubic crystals with an average size of 16 nm that contain silver and chlorine with various functional groups. The synthesized Ag/AgCl-NPs inhibited the growth of three pathogenic bacteria (Bacillus subtilis, Shigella boydii, and Escherichia coli) and two fungi (Aspergillus niger and Trichoderma spp.). Ag/AgCl-NPs inhibited the growth of MCF-7 and EAC cells with the IC50 values of 28 and 84 μg/mL, respectively. No colony was formed in MCF-7 cells in the presence of these nanoparticles as compared with control. Ag/AgCl-NPs induced apoptosis and generated ROS in MCF-7 cells. The expression level of FAS, FADD, and caspase-8 genes increased several folds with the decrease of PARP gene expression. These results demonstrated that the anti-proliferation activity of Ag/AgCl-NPs against MCF-7 cells resulted through ROS generation and induction of apoptosis through the Fas-mediated pathway.

Lithium sulfur batteries (LSBs) are promising next-generation rechargeable batteries due to the high gravimetric energy, low cost, abundance, nontoxicity, and high sustainability of sulfur. However, the dissolution of high-order polysulfide in electrolytes and low Coulombic efficiency of Li anode require excess electrolytes and Li metal, which significantly reduce the energy density of LSBs. Quasi-solid-state LSBs, where sulfur is encapsulated in the micropores of carbon matrix and sealed by solid electrolyte interphase, can operate under lean electrolyte conditions, but a low sulfur loading in carbon matrix (

Solar thermal-driven thermochemical H2O and CO2 splitting offers a carbon-neutral path to produce feedstocks for synthetic fuel production such as hydrogen or synthesis gas. This paper assesses research outcomes for perovskite materials in two-step thermochemical cycles. Experimental, computational and thermodynamic studies are summarized and critically discussed, identifying key attributes for future research. In addition to the critical review, a fast method for the classification of effective thermochemical properties (oxygen vacancy formation enthalpy and entropy) in a wide range of operational temperatures is provided. These properties together with a high-grade of sintering resistance and fast kinetics are the main characteristics required to maximize the solar-to-fuel efficiency of the process. The discovery of optimum material compositions for this application could be effectively achieved by a combination of machine learning, DFT, experimental testing and system modelling, and will require an extensive international research effort. If successful, this could lead to the ultimate development and practical application of thermochemical cycles for fuel production.

This work reports large-area fabrication of polymer [polydimethylsiloxane (PDMS), polycaprolactone (PCL) and polytetrafluoroethylene (PTFE)]-based superhydrophobic surfaces decorated with Cu(OH)2 nanowires. The process flow involves fabrication of nanostructured copper hydroxide surface (Cu(OH)2_NSS) by etching and coating the Cu(OH)2_NSS with a hydrophobic silane layer. This is followed by transferring the nanowires on to the polymer surface. The fabricated surfaces [which are Cu(OH)2 nanowires on PDMS: PDMS_Cu(OH)2, Cu(OH)2 nanowires on PCL: PCL_Cu(OH)2 and Cu(OH)2 nanowires on PTFE: PTFE_Cu(OH)2] exhibited dual scale roughness imparting very good water repelling behavior with a static contact angle greater than 150° and contact angle hysteresis of less than 10°. In addition to water repelling behavior, the fabricated surfaces exhibited bloodphobic behavior making them suitable for specific healthcare applications. Using the same technique, fabrication of superhydrophobic surface with complex 3D shape was also demonstrated. The same methodology was further adopted to fabricate a large-area (22 cm × 22 cm) superhydrophobic surface by replacing silane with stearic acid, which is a non-fluorinated surface modifying agent. This helped in reducing the cost and time of fabrication which is important when it comes to large-scale manufacturing.

Background Several new treatment options have been approved for relapsed and/or refractory multiple myeloma (RRMM). In this systematic review, associations of the efficacy of each approved regimen with adverse events (AEs) and the total cost per cycle were compared with a Bayesian network meta-analysis (NMA) of phase 3 randomized controlled trials (RCTs). Methods Scopus, Cochrane, PubMed Publisher, and Web of Science were searched from January 1999 to July 2018 for phase 3 RCTs of regimens (approved by the US Food and Drug Administration) used in RRMM. The relative ranking of agents was assessed with surface under the cumulative ranking (SUCRA) probabilities. The primary efficacy, safety, and cost outcomes were progression-free survival with the regimen, grade 3 to 4 AEs, and the total cost per cycle (regimen cost plus average cost of managing AEs). Results Fifteen studies including 7718 patients and evaluating 14 different regimens were identified. Daratumumab, lenalidomide, and dexamethasone were ranked highest for reducing progression (hazard ratio, 0.13; 95% credible interval, 0.09-0.19; SUCRA, 1) but carried the highest probability of total cost per cycle ($41,420; 95% Credible Interval [CrCl], $58,665-$78,041; SUCRA, 0.02). Panobinostat, bortezomib, and dexamethasone were the least effective and least safe (SUCRA, 0.24), whereas bortezomib, thalidomide, and dexamethasone emerged as least effective with the highest total cost per cycle (SUCRA, 0.33). Carfilzomib and dexamethasone emerged as the winner when this regimen was considered in terms of efficacy and safety (SUCRA, 0.61) and efficacy and total cost per cycle (SUCRA, 0.60). Conclusions The results of this NMA can provide additional guidance for the decision-making process when one is choosing the most appropriate regimen for RRMM.

Aluminium hybrid metal matrix nano composite (AHMNC) material has all the advantages of conventional metal matrix nano composite with multifunctional properties because of the hybrid reinforcement. As a result, these materials offer a unique combination of properties that are not possible to achieve in conventional materials. The present study deals with the fabrication of aluminium hybrid nano composites by reinforcing Al2014 with nano sized B4C and graphite, processed through stir casting route followed by ultrasonic and squeeze casting. The addition of B4C improves the hardness, whereas Gr particles improve wear resistance in the composite. These combinations of the reinforcements are chosen for application under high abrasive conditions. Ultrasonic agitation has been done to uniformly distribute the reinforcement, whereas squeeze casting is done to reduce the porosity of the cast. The cast composite has been cut to prepare specimens for hardness, tensile strength, and microstructure as per the ASTM standards. The mechanical properties of the composite were evaluated and compared to the conventional. The microstructure of the composites was studied using optical microscopy to investigate the proper distribution of reinforcement and microstructure in the AHMNCs. The result indicates a significant improvement in the hardness. Further, the microstructure studies reveal that the reinforcement was fairly uniformly distributed over the matrix material.

Bacterial tyrosine kinases (BY-kinases) and shikimate kinases (SKs) comprise two structurally divergent P-loop containing enzyme families that share similar catalytic site geometries, most notably with respect to their Walker-A, Walker-B, and DxD motifs. We had previously demonstrated that in BY-kinases, a specific interaction between the Walker-A and Walker-B motifs, driven by the conserved “catalytic” lysine housed on the former, leads to a conformation that is unable to efficiently coordinate Mg2+•ATP and is therefore incapable of chemistry. Here, using enhanced sampling molecular dynamics simulations, we demonstrate that structurally similar interactions between the Walker-A and Walker-B motifs, also mediated by the catalytic lysine, stabilize a state in SKs that deviates significantly from one that is necessary for the optimal coordination of Mg2+•ATP. This structural role of the Walker-A lysine is a general feature in SKs and is found to be present in members that encode a Walker-B sequence characteristic of the family (Coxiella burnetii SK), and in those that do not (Mycobacterium tuberculosis SK). Thus, the structural role of the Walker-A lysine in stabilizing an inactive state, distinct from its catalytic function, is conserved between two distantly related P-loop containing kinase families, the SKs and the BY-kinases. The universal conservation of this element, and of the key characteristics of its associated interaction partners within the Walker motifs of P-loop containing enzymes, suggests that this structural role of the Walker-A lysine is perhaps a widely deployed regulatory mechanism within this ancient family.

Objectives The aim of the present work was to prepare QbD enabled optimization, and to improve the oral bioavailability of freeze-dried polymeric nanoparticles of cinacalcet hydrochloride manufactured by nanoprecipitation and ultrasonication methods using polymers PLGA, and poloxamer-188. Materials and methods The initial screening and optimization were carried out for the formulations by employing Taguchi and Box-Behnken Designs. The FT-IR and DSC revealed no interactions and had no incompatibility among the selected drug and polymers. The nanoparticles were characterized for % drug release, particle size analysis, zeta potential, PDI, SEM, TEM, P-XRD, TGA, DTA, in vitro, and in vivo drug release study. Results In vitro drug release study showed sustained release of the drug from the optimized batch by diffusion mechanism. The optimized nanoparticle formulation was recognized by numerical and graphical methods using validation of the experimental model. The optimized batch was stable as per the ICH stability guidelines for 6 months with no considerable alternation noticed in particle size, entrapment efficiency, and in vitro drug release. The pharmacokinetic parameters of AUC and Cmax data for the optimized formulation increased 3- and 2.9-folds compared to the pure-drug suspension. Conclusion The prepared polymeric nanoparticles formulation is an alternative delivery system for enhanced therapeutic efficacy and bioavailability potential of a model drug to manage long-term normocalcemia in patients with preliminary hyperparathyroidism.

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli ΔspeG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

Prostate cancer (PC) is the second most common cancer in men worldwide. Due to the large-scale sequencing efforts, there is currently a better understanding of the genomic landscape of PC. The identification of defects in DNA repair genes has led to clinical studies that provide a strong rationale for developing poly (ADP-ribose) polymerase (PARP) inhibitors and DNA-damaging agents in this molecularly defined subset of patients. The identification of molecularly defined subgroups of patients has also other clinical implications; for example, we now know that carriers of breast cancer 2 (BRCA2) pathogenic sequence variants (PSVs) have increased levels of serum prostate specific antigen (PSA) at diagnosis, increased proportion of high Gleason tumors, elevated rates of nodal and distant metastases, and high recurrence rate; BRCA2 PSVs confer lower overall survival (OS). Distinct tumor PSV, methylation, and expression patterns have been identified in BRCA2 compared with non-BRCA2 mutant prostate tumors. Several DNA damage response and repair (DDR)-targeting agents are currently being evaluated either as single agents or in combination in patients with PC. In this review article, we highlight the biology and clinical implications of deleterious inherited or acquired DNA repair pathway aberrations in PC and offer an overview of new agents being developed for the treatment of PC.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic’s efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

Aqueous electrochemical systems suffer from a low energy density due to a small voltage window of water (1.23 V). Using thicker electrodes to increase the energy density and highly concentrated “water-in-salt” (WIS) electrolytes to extend the voltage range can be a promising solution. However, thicker electrodes produce longer diffusion pathways across the electrode. The highly concentrated salts in WIS electrolytes alter the physicochemical properties which determine the transport behaviors of electrolytes. Understanding how these factors interplay to drive complex transport phenomena in WIS batteries with thick electrodes via deterministic analysis on the rate-limiting factors and kinetics is critical to enhance the rate-performance in these batteries. In this work, a multimodal approach—Raman tomography, operando X-ray diffraction refinement, and synchrotron X-ray 3D spectroscopic imaging—was used to investigate the chemical heterogeneity in LiV3O8–LiMn2O4 WIS batteries with thick porous electrodes cycled under different rates. The multimodal results indicate that the ionic diffusion in the electrolyte is the primary rate-limiting factor. This study highlights the importance of fundamentally understanding the electrochemically coupled transport phenomena in determining the rate-limiting factor of thick porous WIS batteries, thus leading to a design strategy for 3D morphology of thick electrodes for high-rate-performance aqueous batteries., Multimodal Raman and synchrotron X-ray analysis reveals that the rate-limiting factor of thick porous LiMn2O4 electrodes in a water-in-salt electrolyte is the ionic diffusion in the liquid phase. The finding furthers the understanding of kinetics in an aqueous system for electrochemical energy storage with highly concentrated electrolytes, guiding the future design of advanced 3D-architecture electrodes.

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by d-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields.

Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca(2+) thereon. We find that in the absence of Ca(2+), CaM exclusively utilizes its C‐terminal lobe (CaM(C)) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca(2+)‐loaded N‐terminal lobe of CaM (CaM(N)) at increased Ca(2+) levels serves to enhance the affinity further. These latter interactions under Ca(2+) saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca(2+)‐free state. Our data are consistent with a scenario in which Ca(2+) enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.

Developing novel and more efficient filters for chemical warfare agent (CWA) decomposition remains an important challenge for modern technology due to the continuous threat those weapons present in...

Drug substance (DS) appearance is an important quality attribute that is tested before product release. Color variation in DS batches or color change during storage can be indicative of the presence of contaminants, impurities or degradation products and may be linked to the manufacturing process, DS formulation or the stability of the therapeutic proteins. Atypical DS color other than colorless or pale yellow has been recently observed and may result in rejected manufacturing batches and financial loss due to concerns on product quality and visually matching placebo in clinical trials. This review focuses on the origins of atypical color and changes in DS during manufacturing processes using mammalian cell culture and storage. The purposes here are to understand the link between changes in the DS color and various process parameters and to provide necessary information to our readers regarding product quality control strategies with respect to DS color.

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers., The ERK signalling pathway is activated in many cancers, however ERK1 and ERK2 are difficult to target pharmacologically. Here, the authors identify a small molecule inhibitor that binds covalently to the D-recruitment site of ERK and induces cell death and reduces tumour growth in mice.

Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K(562–725) that encodes two α-helical repeats. The structure of eEF-2K(562–725) shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Further, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K(562–725). We find that eEF-2K(562–725) engages eEF-2 with an affinity comparable to that of the full-length enzyme. Further, eEF-2K(562–725) is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K(562–725) encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36-40 g/m2 ) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m2 , similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure–activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507–1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507–8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507–8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein–protein interactions.

Depth filtration is a commonly-used bioprocessing unit operation for harvest clarification that reduces the levels of process- and product-related impurities such as cell debris, host-cell proteins, nucleic acids and protein aggregates. Since depth filters comprise multiple components, different functionalities may contribute to such retention, making the mechanisms by which different impurities are removed difficult to decouple. Here we probe the mechanisms by which double-stranded DNA (dsDNA) is retained on depth filter media by visualizing the distribution of fluorescently-labeled retained DNA on spent depth filter discs using confocal fluorescence microscopy. The extent of DNA displacement into the depth filter was found to increase with decreasing DNA length with increasing operational parameters such as wash volume and buffer ionic strength. Finally, using 5ethynyl-2'-deoxyuridine (EdU) to label DNA in dividing CHO cells, we showed that Chinese hamster ovary (CHO) cellular DNA in the lysate supernatant migrates deeper into the depth filter than the lysate re-suspended pellet, elucidating the role of the size of the DNA in its form as an impurity. Apart from aiding DNA purification and removal, our experimental approaches and findings can be leveraged in studying the transport and retention of nucleic acids and other impurities on depth filters at a small scale.

Background To date, there is no country-representative study on tobacco and alcohol use in Zambia and Zimbabwe despite the fact that these two countries rank among the top producers of tobacco worldwide. To fill this research gap, we conducted this study to measure the prevalence of tobacco and alcohol consumption among adolescent and adult men in Zambia and Zimbabwe. Special attention was given to the age differential in the prevalence of smoking and drinking. Methods Country-representative data on alcohol and tobacco use along with relevant sociodemographic parameters were collected from Demographic and Health Surveys. Sample population were 23,169 men (14,773 from Zambia and 8,396 from Zimbabwe) aged 15-54 years. Data were analysed using multivariate regression techniques. Results Prevalence of tobacco use was 19.9% (19.0-20.9) in Zambia and 18.4% (17.4-19.5) in Zimbabwe, and that of alcohol was 37.6% (36.4-38.9) in Zambia and 50.5 (48.9-52.1) in Zimbabwe. In both of the countries, the majority of the tobacco and alcohol users were aged between 24-39 years. Results of multivariate regression analysis showed a significant positive association between tobacco and alcohol use with age, place of residence, religious affiliation, marital status, education and wealth quintile. Conclusion Nearly one-fifth of all men in the age group of 15-54 years smoke tobacco in Zambia and Zimbabwe, with the prevalence being most pronounced among those aged between 25-39 years. The predominantly young age structure of alcohol and tobacco users warrant demographically tailored anti-tobacco and alcohol controlling programmes.

Phosphate solubilizing bacteria (PSB) that can withstand high cadmium (Cd) stress is a desired combination for bioremediation. This study evaluated the Cd bioremediation potential of four PSB strains isolated from the contaminated soils of a municipal solid waste (MSW) discarding site (Guwahati, India). PSB strains were cultured in Pikovskaya (PVK) media, which led to higher acid phosphatase (ACP) activity and the release of organic acid. Optical density (OD) measurements were performed to determine the growth pattern of PSB; furthermore, Cd uptake by PSB was evaluated using infrared spectroscopy (IR) and X-Ray Diffraction (XRD) analyses. The 16S rRNA taxonomic analysis revealed that all the four promising PSB strains belonged to either Bacillus sp. or Enterobacter sp. One strain (SM_SS8) demonstrated higher tolerance towards Cd (up to 100 mg L−1). Flow cytometry analysis revealed 70.92%, 46.93% and 20.4% viability of SM_SS8 in 10, 50 and 100 mg L−1, respectively in PVK media containing Cd. This study has therefore substantiated the bioremediation of Cd from polluted soil by the PSB isolates. Thus, experimental results revealed a potential combo benefit, phosphate solubilization along with Cd remediation.

BACKGROUND Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.

Bevacizumab, a monoclonal immunoglobulin-G1 antibody directed against vascular endothelial growth factor (VEGF), inhibits angiogenesis. Gastrointestinal perforation is a serious and often fatal adverse event related to bevacizumab use. Bevacizumab is indicated in the treatment of colorectal malignancies, certain subtypes of non-small cell lung carcinoma, metastatic renal cell carcinomas, and cervical cancers. It is also indicated in the treatment of recurrent glioblastoma (GBM) in adult patients as the sole treatment agent or in combination with other antineoplastic medications. We present a case of a patient on bevacizumab currently with glioblastoma multiforme and seizures, who was previously treated with radiation treatment and temozolomide. The patient presented to the emergency room with abdominal pain, seizures and was diagnosed to have an intestinal perforation.

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.

This paper describes a simplified affinity precipitation process for the purification of mAbs from complex mixtures using elastin-like polypeptide fused to a single Z domain of protein A (ELP-Z). This approach eliminates several steps in the original process by directly extracting the mAb from the affinity precipitate, without the need for resolubilization. The efficacy of this elution without resolubilization (EWR) approach for obtaining pure mAb is demonstrated and the effects of mixing are examined. This simplification of the affinity precipitation process may facilitate the implementation of ELP-Z based mAb bioprocessing, particularly in a continuous scenario.